Abstract While current efforts in the analysis of neural circuits focus on interneuronal connectivity mediated by chemical synapses, less is known about the contribution of electrical synapses. Electrical transmission is mediated by neuronal gap junctions, which are widely distributed throughout the vertebrate brain. However, the extent and subcellular distribution of electrical synapses within neural circuits has been difficult to assess because: 1) antibodies targeting connexins (gap junction forming proteins) vary in their specificity, resulting in false positive or negative staining, and therefore potentially generating wrong or incomplete maps of connectivity, and 2) current electron microscopy protocols used to generate connectomes are unfavorable for detecting gap junctions, thus biasing the description of neuronal interconnection to chemical synapses. To overcome this problem, we propose to develop transgenic-based methods that will allow investigating the presence and contribution of electrical synapses in zebrafish, a model organism that has been identified as particularly advantageous for the analysis of neural circuits by the Brain Initiative. More specifically, we propose to create a Library of Transgenic Zebrafish to study Electrical Synaptic Transmission which will make it possible to generate, for the first time, a complete map of the distribution of electrical synapses in a vertebrate nervous system. The proposal involves generating three types of fish at which connexins and/or its promoters are tagged with fluorescent proteins or functional sensors that, combined, will allow comprehensive examination of the functional contributions of electrical synapses to circuits underlying various behaviors with cell specificity. Aim 1 is to generate transgenic zebrafish at which the promoters of neuronal connexins are linked to reporter fluorescent proteins. The availability of these animals will allow for the establishment of the presence of a particular gap junction protein in a cell or circuit of interest, a notoriously challenging problem, as cells expressing a particular connexin will be fluorescently labeled. Aim 2 is to generate transgenic zebrafish at which zebrafish neuronal connexins are tagged with fluorescent proteins. We will engineer the endogenous neuronal connexin proteins with fluorescent proteins or affinity tags to assess the number and subcellular location of electrical synapses of a cell with its connected neighbors. Because of the design of the constructs, tagged connexins can be imaged by diverse methods including single or 2-photon imaging of living animals or tissues, or chemical enhancements suitable for electron microscopic analysis. Finally, Aim 3 is to generate transgenic zebrafish to study functional contributions of electrical synapses to neuronal circuits. We propose to generate transgenic fish in which neuronal connexins are linked to Ca++ sensors that will make possible detecting active electrical synapses as well those undergoing plastic changes. The proposed approach represents a significant improvement over current methods of analysis and, if successful for analysis of zebrafish neural circuits, could be potentially applied to analysis of electrical transmission in mammalian species.